Pulsed contractions of an actin–myosin network drive apical constriction

Abstract

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis1 , 2 . Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and Non-Muscle Myosin-II (myosin) belt underlying adherens junctions 3-7. However, it is unclear whether other force-generating mechanisms can drive this process. Here, we use real-time imaging and quantitative image analysis of Drosophila gastrulation to show that apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighboring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inward. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions while expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled sub-cellular ratchet to incrementally reduce apical area.During Drosophila gastrulation, apical constriction of ventral cells facilitates the formation of a ventral furrow and the subsequent internalization of the presumptive mesoderm. Although myosin is known to localize to the apical cortex of constricting ventral furrow cells [8][9][10][11] , how myosin produces force to drive constriction is not known. Understanding this mechanism requires a quantitative analysis of cell and cytoskeletal dynamics. Therefore, we developed methods to visualize and quantify apical cell shape using Spider-GFP, a GFP-tagged transmembrane protein that outlines individual cells (Fig. 1a, 1b , Supplemental Figure 1 , Video 1) 12 . Ventral cells constricted to ~50 % of their initial apical area before the onset of invagination and continued to constrict during invagination (Fig. 1c, 1e). Although the average apical area steadily decreased at a rate of ~5 μm 2 /min, individual cells exhibited transient pulses of rapid constriction that exceeded 10-15 μm 2 /min (Fig. 1d, 1f, 1g, and Video 2). During the initial 2 minutes of constriction, weak constriction pulses were often interrupted by periods of cell stretching. However, at 2 minutes constriction pulses increased in magnitude and cell shape

Assessing the critical period for Rho kinase activity during Drosophila ventral furrow formation

“…This initial constriction phase will be referred to as ''random apical constriction'' (RAC). These constriction events are reversible, as some of the constricted cells relax their apices (Kam et al, 1991;Oda and Tsukita, 2001;Martin et al, 2009). This period is thought to be a preparative early phase, possibly leading to, or coincident with, apical flattening (Kam et al, 1991;Martin et al, 2009).…”

“…These constriction events are reversible, as some of the constricted cells relax their apices (Kam et al, 1991;Oda and Tsukita, 2001;Martin et al, 2009). This period is thought to be a preparative early phase, possibly leading to, or coincident with, apical flattening (Kam et al, 1991;Martin et al, 2009). The second phase begins when cells constrict their apices in a rapid, concerted manner (Kam et al, 1991;Oda and Tsukita, 2001;Martin et al, 2009).…”

“…This period is thought to be a preparative early phase, possibly leading to, or coincident with, apical flattening (Kam et al, 1991;Martin et al, 2009). The second phase begins when cells constrict their apices in a rapid, concerted manner (Kam et al, 1991;Oda and Tsukita, 2001;Martin et al, 2009). This fast constriction phase will be referred to as ''concerted apical constriction'' (CAC).…”

“…It is currently believed that constriction of an actomyosin medial meshwork generates the force required to drive all of the cell-shape changes in a coordinated manner (Martin et al, 2009). However, it has not been possible to genetically perturb the actomyosin cytoskeleton, or more specifically myosin II, to study the role of myosin II in VFF because of its essential role during oogenesis and the final step of cellularization, both of which precede VFF (Wheatley et al, 1995;Royou et al, 2004).…”

Optogenetic control of morphogenesis goes 3D

“…The Drosophila ventral furrow has since become an excellent model system for understanding how epithelial folding can occur, with numerous studies demonstrating the importance of apical actomyosin‐driven constriction in driving indentation of the epithelium (Leptin & Grunewald, 1990; Sweeton et al , 1991; Dawes‐Hoang et al , 2005; Martin et al , 2009). Thanks to these impressive studies, it is often thought that the problem of ventral furrow formation is now solved.…”